The invention relates generally to methods and compositions for use in production of GABAergic cells.
The vast majority of neurons in the forebrain use either glutamate or gamma aminobutyric acid (GABA) as a neurotransmitter. Whereas glutamate sends excitatory signals, GABA sends inhibitory signals in the adult brain. In the cerebral cortex, projection neurons (neurons that project their axons to distant targets) are glutaminergic whereas local circuit neurons (interneurons) are GABAergic. Imbalances in the function of these physiologically antagonistic cell types can lead to disorders such as epilepsy, which is generally due to excessive excitatory activity. Epilepsy can be treated with medicines that mimic the function of GABAergic cells.
Unlike the cerebral cortex, most projection neurons of the basal ganglia (striatum and globus pallidus) are GABAergic. Disorders such as Huntington""s disease lead to the degeneration of these neurons. Despite their large numbers and essential functions, little is known about the specification and differentiation of GABAergic neurons of the forebrain, or the precise genetic mechanisms regulating these phenomena.
One family of genes, the Dlx homeobox genes, has attracted interest due to their patterns of expression in the forebrain during development. Early in gestation, Dlx gene expression in the telencephalon is restricted to the primordia of the basal ganglia, and is excluded from the cerebral cortex (Porteus et al. (1991) Neuron 7:221-229; Bufone et al. (1993) J. Neurosci. 13(7):3155-3172), where its expression is co-extensive with cells producing GABA (Anderson et al. (1997) Neuron 19:27-37). Then, beginning around E12.5, Dlx+/GABA+ cells are found migrating along two tangential pathways that introduce these cells into cortical regions of the telencephalon: a lateral and a medial pathway. The lateral migratory pathway originates in both the lateral and medial ganglionic eminences of the basal ganglia and introduces specific types of Dlx+/GABAergic interneurons in the striatum, olfactory cortex, neocortex and hippocampus (Porteus et al. (1994) J. Neurosci. 14(11):6370-6383; Anderson et al. (1997) Science 278:474-476). Mice lacking Dlx1 and Dlx2 have a four-fold reduction in the numbers of GABAergic neocortical neurons (Anderson et al. (1997) Science 278:474-476). Others have also identified this lateral pathway, but had not demonstrated GABAergic interneurons in this pathway prior to our discovery (deCarlos et al. (1996) J. Neurosci. 16:6146-6156; Tamamaki et al. (1997) J. Neurosci. 17:8313-8323). Since then other groups have also reported migration along the lateral pathway (Lavadas et al. (1999) J. Neurosci. 19:7881-7888; Wichterle et al. (1999) Nat. Neurosci. 2:461-466.
The medial migratory pathway (also known as the rostral migratory stream), appears to originate in the region of the lateral ganglionic eminence and septum, and is the source for GABAergic interneurons of the olfactory bulb and perhaps subsets of cortical interneurons. See, Gadisseux et al. (1992) J. Comparative Neurol. 324:94-114; Luskin (1993) Neuron 11:173-189); DeDiego et al. (1994) Eur. J. Neurosci. 6:983-997; Lois and Alvarez-Buylla (1994) Science 271:264:1145-1148; and Meyer et al. (1998) J. Comp. Neurol. 397:493-518. This pathway contains Dlx+ cells (Porteus et al. (1994) J. Neurosci. 14(11):6370-6383); Dlx1 and Dlx2 mutants have a  greater than 95% reduction in the number of GABAergic neurons of the olfactory bulb (Bulfone et al. (1998) Neuron 21:1273-1282).
The lateral pathway, and perhaps the medial as well, seed the proliferative zone (subventricular zone) of the postnatal rodent brain with Dlx+ cells. This proliferative zone is known to be the source of postnatal neurogenesis. In particular, this proliferative zone is a source of GABAergic interneurons of the olfactory bulb (Luskin (1993) Neuron 11:173-189; and Lois and Alvarez-Buylla, (1994) Science 264:1145-1148).
The identity and relationship of the Dlx gene family members are just beginning to be elucidated. To date, six mammalian Dlx genes have been identified, of which four (Dlx1, Dlx2, Dlx5 and Dlx6) are expressed in the central nervous system (CNS). Within the CNS they appear to be exclusively expressed in the forebrain, where they are expressed in most, if not all, developing GABAergic cells. See, Porteus et al. (1991) Neuron 7:221-229; Price et al. (1991) Nature 351:748-751; Bulfone et al. (1993) Mech. Dev. 40(3):187; Simeone et al. (1994) Proc. Natl. Acad. Sci. USA 91:2250-2254; Stock et al. (1996) Proc. Natl. Acad. Sci. USA 93:10858-10863; Liu et al. (1997) Dev. Dyn. 210:498-512; Anderson et al. (1997) Neuron 19:27-37; Anderson et al. (1997) Science 278:474-476; Bulfone et al. (1998) Neuron 21:1273-1282; and Anderson et al. (1999) Cereb. Cortex 6:646-54.
Expression of the Dlx genes generally occurs in a temporal sequence during differentiation of GABAergic cells, with expression of these genes being at least somewhat overlapping. Dlx1 and Dlx2 are expressed first, in mitotically active progenitor cells within the ventricular zone; expression of Dlx1 and Dlx2 is followed by Dlx5, and finally Dlx6 (Liu et al. (1997) Dev. Dyn. 210:498-512). This temporal relationship implies a regulatory cascade in which the early expressing Dlx genes regulate the expression of later expressed Dlx genes.
Evidence of a Dlx regulatory cascade is supported by observations of Dlx expression in transgenic mice lacking distinct Dlx genes. For example, in the mice lacking Dlx1 and Dlx2 expression (transgenic xe2x80x9cknock-outxe2x80x9d mice), Dlx5 and Dlx6 expression is greatly attenuated in the forebrain (Anderson et al. (1997) Neuron 19:27-37). Studies with the Dlx1/Dlx2 transgenic knockout mice also indicate at least some degree of functional redundancy between the Dlx genes, further complicating the study of this complex gene family. For example, mice carrying single mutations in either Dlx1 or Dlx2 have very mild defects in their forebrain, whereas the Dlx1/Dlx2 double mutant mice have a severe block in the differentiation of basal forebrain GABAergic neurons in the basal forebrain and basal ganglia (Anderson et al. (1997) Neuron 19:27-37). These transgenic animal studies have supported the notion that development of GABAergic forebrain cells depends on the function of the Dlx genes: roughly 80% of neocortical, and greater than 95% of olfactory bulb and hippocampal GABAergic interneurons do not fully develop in Dlx1/Dlx2 double mutant transgenic mice (Anderson et al. (1997) Science 278:474-476; and Bulfone et al. (1998) Neuron 21:1273-1282).
Recent evidence shows that neurotransmitter subtype specification is linked to dorsoventral patterning (Marin et al. (2000) J. Neurosci. 20:6063-6076; and Wilson and Rubenstein, (2000) Neuron 28:641-651). For example, in the ventral spinal cord, longitudinal progenitor domains, that are arrayed as distinct dorsoventral tiers, give rise to distinct types of neurons: cholinergic motor neurons arise from more ventral positions than most GABAergic interneurons (Briscoe et al. (2000) Cell 101:435-445; and Sander et al. (2000) Genes Dev. 14:2134-2139). A similar organization appears to be found in the embryonic telencephalon. In both of these tissues, Nkx genes regulate specification of cholinergic neurons (Sussel et al. (1999) Development 126:3359-3370). Transcription factors that are expressed in progenitor domains abutting GABAergic neuronal zones are candidates for regulating GABAergic specification.
In the telencephalon, members of the Dlx homeobox gene family are expressed in, and regulate the development of the primordia of the basal ganglia (Anderson et al. (1997) Neuron 19:27-37). These large nuclei consist of GABAergic projection neurons. In addition, the Dlx genes are expressed in, and regulate the development of, neurons that tangentially migrate from the basal telencephalon into the cerebral cortex (Anderson et al. (1997) Science 278:474-476; and Pleasure et al. (2000) Neuron 28:727-740). These neurons give rise to GABAergic interneurons of the cerebral cortex, hippocampus and olfactory bulb (Bulfone et al. (1998) Neuron 21:1273-1282; and Lavdas et al. (1999) J. Neurosci. 19:7881-7888). The cortical GABAergic and glutamatergic neurons arise from distinct telencephalic progenitor zones and are under distinct genetic controls (Anderson et al. (1997) Science 278:474-476; Parnavelas, J. G. (2000) Trends Neurosci. 23:126-131; and Hevner et al. (2001) Neuron 29:353-366).
The molecular mechanisms through which the Dlx genes regulate the development of forebrain GABAergic neurons remain to be elucidated. As is evident from the above, while great strides have been made in understanding the production of GABAergic neurons, no one line of evidence has pointed to a distinct single gene or set of genes that can guide an immature cell toward development into a GABAergic cell. Without a simple, elegant system to trigger development of GABAergic neurons, there is little hope for the production of GABAergic cells in vitro in numbers that render the cells useful in, for example, screening assays to identify new drugs that regulate interneuronal activity or as sources of cells for replacement therapy. The present invention addresses this problem.
The invention features methods and compositions for the production of GABAergic cells, particularly GABAergic neurons. Production of GABAergic cells is accomplished by increasing activity of a Dlx gene (e.g., DLX1, DLX2, or DLX5) in an immature neuronal cell. The increase in Dlx activity causes differentiation of the immature neuronal cell into a neuronal cell exhibiting the GABAergic phenotype. The invention also encompasses use of GABAergic cells produced by the method of the invention in, for example, identification of agents that affect GABAergic cell activity and survival, and in replacement therapy.